A Methyl Scan of the Pyrrolidinium Ring of Nicotine Reveals Significant Differences in Its Interactions with a7 and a4b2 Nicotinic Acetylcholine Receptors

Article abstract of DOI:10.1124/MOL.119.118786

The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the a4b2 and a7 subtypes. A "methyl scan" of the pyrrolidinium ring was used to detect differences in nicotine's interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in nicotine's receptor interactions. Replacing the 19-N-methyl with an ethyl group or adding a second 19-N-methyl group significantly reduced interaction with a4b2 but not a7 receptors. The 29-methylation uniquely enhanced binding and agonist potency at a7 receptors. Although 39- A nd 59-trans-methylations were much better tolerated by a7 receptors than a4b2 receptors, 49-methylation decreased potency and efficacy at a7 receptors much more than at a4b2 receptors. Whereas cis-59-methylnicotine lacked agonist activity and displayed a low affinity at both receptors, trans-59-methylnicotine retained considerable a7 receptor activity. Differences between the two 59-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine analog A84543 were consistent with what was found for the 59-methylnicotines. Computer docking of the methylnicotines to the Lymnaea acetylcholine binding protein crystal structure containing two persistent waters predicted most of the changes in receptor affinity that were observed with methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller effects of 19-, 39-, and 59-methylations and the greater effects of 29- A nd 49-methylations on nicotine a7 nAChR interaction might be exploited for the design of new drugs based on the nicotine scaffold.

Full text of DOI:10.1124/MOL.119.118786

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A Methyl Scan of the Pyrrolidinium Ring of Nicotine Reveals Significant Differences in its
Interactions with α7 and α4β2 Nicotinic Acetylcholine Receptors
Hong Xing, Kristin W. Andrud, Ferenc Soti, Anne Rouchaud, Stephan C. Jahn, Ziang Lu, Yeh-
Hyon Cho, Sophia Habibi, Patrick Corsino, Svetoslav Slavov, James R. Rocca, Jon M.
Lindstrom, Ron J. Lukas and William R. Kem*
Department of Pharmacology and Therapeutics, College of Medicine, University of Florida,
Gainesville, FL 32610, USA (HX, KWA, FS, AR, SH, SJ, LZ, Y-HC, PC, WRK); National
Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079
(SS); AMRIS, McKnight Brain Institute, University of Florida, (JRR); Department of
Neuroscience, University of Pennsylvania, Philadelphia, PA 19104 (JML); Division of
Neurobiology, Barrow Neurological Institute, Phoenix, AZ 85013 (RJL); Present Address: Dept.
of Biological Sciences, University of Denver, Denver, CO 80208 (KWA)

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Disclaimer: The views presented in this article are those of the authors and do not necessarily
reflect those of the U.S. Food and Drug Administration. No official endorsement is intended nor
should be inferred.
Key Words: Acetylcholine, Drug Design, Methyl Scan, Neurodegenerative Disease,
Nicotine, Nicotinic Acetylcholine Receptor, Schizophrenia, Smoking Cessation

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Running title: Methyl scan of the nicotine pyrrolidinium ring
*To Whom Correspondence Should Be Addressed
Name: William R. Kem
Phone: 352-392-0669
FAX: 352-392-9696
Email: wrkem@ufl.edu
Address: Department of Pharmacology and Therapeutics
University of Florida
P.O. Box 100267
Gainesville, FL 32610-0267
Number of text pages: 32
Number of tables: 6
Number of figures: 4
Number of references: 61
Number of words in Abstract: 232
Number of words in Introduction: 935
Number of words in Discussion: 1791
Abbreviations: AChBP, acetylcholine binding protein; α-BTX, α-
bungarotoxin; nAChR, nicotinic acetylcholine receptor

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ABSTRACT
The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the α4β2 and α7
subtypes. A “methyl scan” of the pyrrolidinium ring was used to detect differences in nicotine’s
interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp
and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in
nicotine’s receptor interactions. Replacing the 1’-N methyl with an ethyl group or adding a
second 1’-N methyl group significantly reduced interaction with α4β2 but not α7 receptors. 2’-
methylation uniquely enhanced binding and agonist potency at α7 receptors. Although 3’and
5’trans-methylations were much better tolerated by α7 receptors than α4β2 receptors, 4’
methylation decreased potency and efficacy at α7 receptors much more than at α4β2 receptors.
While cis-5’-methylnicotine lacked agonist activity and displayed a low affinity at both
receptors, trans-5’methylnicotine retained considerable α7 receptor activity. Differences
between the two 5’-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine
analog A84543 were consistent with what was found for the 5’-methylnicotines. Computer
docking of the methylnicotines to the Lymnaea AChBP crystal structure containing two
persistent waters predicted most of the changes in receptor affinity that were observed with
methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller
effects of 1’-, 3’- and 5’-methylations and the greater effects of 2’- and 4’-methylations on
nicotine α7 nAChR interaction might be exploited for the design of new drugs based on the
nicotine scaffold.

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SIGNIFICANCE
Using a comprehensive “methyl scan” approach we show that the orthosteric binding sites
for acetylcholine and nicotine in the two major brain nicotinic acetylcholine receptors interact
differently with the pyrrolidinium ring of nicotine, and suggest reasons for the higher affinity of
nicotine for the heteromeric receptor. Potential sites for nicotine structure modification were
identified that may be useful in the design of new drugs targeting these receptors.

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INTRODUCTION
Nicotine activates a variety of ligand-gated acetylcholine receptors (nAChRs) that play
important signaling roles in neuronal and some non-neuronal cells and has a number of
potentially therapeutic effects. However, it is one of the most addictive of drugs when self-
administered as a tobacco product. Nicotine is also a widely used as a useful smoking cessation
drug in non-smoked formulations (Prochaska and Benowitz, 2016). Several nicotine analogs
have already been tested as potential treatments for neurodegenerative and psychiatric disorders.
Vertebrate nAChRs are cation permeant ion channels composed of five homologous
subunits. Molecular biological investigations have revealed genes for 17 different subunits, so a
large number of subunit combinations is theoretically possible and >15 pentameric subunit
combinations have already been identified in mammalian tissues (Wu and Lukas, 2011). At least
two α subunits occur in each nAChR pentamer. Agonist binding sites are located within a
groove between the N-terminal extracellular domains of an α subunit and an adjacent subunit.
The α7 and α4β2 nAChRs are the most numerous and widely distributed receptor subtypes in the
brain and have been demonstrated to participate in cognitive function. α7 nAChRs are mainly
homomeric complexes containing five α7 monomers, though small concentrations of α7β2
nAChRs also occur in the brain (Moretti et al., 2014). All the other nAChRs contain two or three
non-α subunits. Significant decreases in the concentrations of these two receptors have been
found in the postmortem brains of Alzheimer’s and Parkinson’s disease patients (Burghaus et al.,
2003). In many schizophrenics, the alpha7 receptor is expressed at lower than normal levels and
this is thought to contribute to deficient sensory gating and cognition (Martin et al., 2004). There
is considerable pharmaceutical interest in developing selective nicotinic agonists and allosteric
modulators that target either α7 or α4β2 nAChRs for treating cognitive deficits in

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neurodegenerative diseases. Partial agonists for α4β2 receptors like varenicline and cytisine are
widely used as smoking cessation drugs. In addition, α7 nAChRs occuring in macrophages (and
their brain counterparts, microglia), lymphocytes, keratinocytes, lung epithelium and certain
cancer cells modulate a variety of signaling pathways and have become attractive therapeutic
targets (Bertrand et al., 2015; Bouzat et al, 2018).
Despite the widespread use of nicotine as an experimental nAChR probe and lead
compound for the design of new drugs, the molecular basis for its preferential interactions with
certain nAChRs is still poorly understood. Initial chemical modification and mutagenesis studies
led to the identification of key aromatic amino acid side chains and peptide bonds constituting
ACh binding sites (Changeux, 2012). A combination of cation-pi, electrostatic, hydrogen and
van der Waals bonding forces are involved in ligand (agonist and competitive antagonist)
recognition (Cashin et al., 2005; Xiu et al., 2009; Puskar et al., 2011). High resolution crystal
structures of several homologous acetylcholine binding proteins (AChBPs) and their analogs
mutated to better resemble the α7 orthosteric binding site have been available for some time
(Celie et al., 2004; Hansen et al., 2005; Li et al., 2011). Crystal structures of the extracellular
portions of the adult skeletal muscle α1 subunit and the α10 subunit binding α-bungarotoxin (α-
BTX) have been reported (Delissanti et al., 2007; Zouridakis et al., 2014). Recently, a crystal
structure of the nearly whole α42β23 nAChR became available (Morales-Perez et al, 2016).
These structures have been very useful for developing homology models of various nAChRs and
have enabled ligand docking studies.
In view of the multiplicity of nAChRs and the need to minimize the adverse effects of
nicotinic drugs on unintended nAChRs, it is desirable to identify structural differences between
their orthosteric binding sites to enable the design of subtype selective drugs. In the present study

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we employ a “methyl scan” (Black et al., 1972) approach to assess the consequences of single
methyl substitutions for each of the eight hydrogens located on the pyrrolidinium ring of
nicotine. Methyl substitution was selected because of its relatively minimal effect (a 16 %
increase) in the mass of the pyrrolidinium moiety. The methyl group, like the four methylene
groups in this ring, does not appreciably affect the lipophilic nature of the ring, introduces no
new hydrogen-bonding atoms and does not diminish ionization of the pyrrolidinium nitrogen,
which has been shown to be essential for efficient nAChR binding by this compound (Barlow
and Hamilton, 1962; Jeng and Cohen, 1980).
The actions of a few alkylated nicotines on isolated tissues and organs have been reported
(Glassco et al., 1994; Dukat et al., 1996). Two laboratories reported extensive rat brain nAChR
(later shown to be largely α42β) binding data on the effect of individual methyl substitutions at 4
of the 8 hydrogens on the pyrrolidinium ring (Lin et al. 1994; Kim et al. 1996; Wang et al.,
1998). In this paper we report a comprehensive functional and radioligand binding analysis of
all the possible chiral nicotine analogs generated by methyl replacement of each H atom attached
to the pyrrolidinium ring of (S)nicotine for α7 as well as α4β2 nAChRs. In addition, we evaluate
the effect of increasing the size of the pre-existing 1’N-methyl group. To test the generality of
our results with nicotine, we extended part of our methyl-scan to a highly potent nicotine analog,
A84543, whose two nicotine-like rings are connected through an oxymethylene bridge that
preserves the chirality of the 2’-pyrrolidinium ring (Abreo et al., 1996). Our results indicate that
the pyrrolidinium moiety in these compounds has a similar binding mode in both nAChR binding
sites but that the α7 site is less sensitive to most methyl substitutions.
[Fig. 1]

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MATERIALS AND METHODS
Nicotine analogs
Preparation of all known compounds followed methods already described in the literature
(See Fig. 1 in Rouchaud and Kem (2012) for a summary of most methods we employed). The
compound 1H and 13C NMR spectral and MS spectrometric data of compounds whose syntheses
were previously reported agreed with the published data. The chiral HPLC conditions for
obtaining the individual enantiomers from the racemic 2’-, 3’- and 5’methylnicotines are
provided in the Supplement. Nicotine dihydrogen tartrate salt (MW 462) was obtained from
Sigma-Aldrich (St. Louis, MO). 1’-Methylnicotinium iodide (MW 304) was purchased from
Toronto Research Chemicals (Toronto, Canada) and its identity was checked by NMR
spectroscopy. In this paper the relative configurations (cis- or trans-) of the pyrrolidinium ring
methyl substituents are always expressed with respect to the configuration of the pyridyl
substituent. All alkylnicotines in Tables 1-5 have the same 2’(S)-pyrrolidinium ring
configuration as in natural (S)nicotine. Data for 3’-methyl and 5’-methyl(R)nicotines are
presented in Table 6.
Functional experiments with Xenopus oocytes
Frogs were purchased, maintained and used under a UF IACUC approval for the Kem
laboratory. Mature female frogs (Xenopus laevis) were anesthetized by immersion in a 1.5 g/L
solution of ethyl 3-aminobenzoate methanesulfonate (MP Biomedicals, Solon, OH) for 30 min.
After the frog was completely unresponsive and immobile it was decapitated with a guillotine
and its spinal cord pithed before excision of the ovary, which was placed into calcium-free Barth
saline (88 mM NaCl, 1 mM KCl, 2.38 mM NaHCO3, 0.82 mM MgSO4, 15 mM HEPES, 0.012

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mg/ml tetracycline hydrochloride (Sigma-Aldrich), pH 7.3±0.1), opened with forceps and
washed three times with saline. Then 50 ml of 1.25 mg/ml type 1 collagenase (Worthington
Biochemical Corporation, Freehold, NJ) solution dissolved in Ca2+-free Barth saline was added
and the oocyte containing mass was gently shaken for 2 hours at room temperature. After
washing three times with calcium-free saline and then three times with Barth saline containing
0.7 mM Ca2+, healthy stage 5 oocytes were transferred into dishes and incubated at 17°C
overnight. Oocytes were routinely injected with 50 nl of a solution containing 20 ng α7 mRNA
or 50 nl of a mixture of α4 and β2 mRNA (10 ng each), using a Drummond Nanoject II Auto-
Injector. In some pilot experiments, oocytes were injected with 4:1 or 1:4 ratios (20 ng mRNA,
total) of the two mRNAs to determine whether nicotine action under our experimental conditions
was sensitive to subunit stoichiometry (Nelson et al., 2003). Finally, in one final set of
experiments intended to investigate only the high agonist sensitivity α42β23 nAChR, an equal
weight (5ng) of RNA for the human β2AGSα4 concatamer was injected with 5 ng β2 RNA in
each oocyte, since this ratio has been shown to minimize expression of alternative stoichometries
of this receptor (Kuryatov et al., 2005;2007). Injected oocytes were cultured in Barth’s saline for
5-10 days at 17°C with daily changes in the saline prior to recordings.
Individual oocytes were placed into a 20 µl oocyte perfusion chamber (Model OPC-1
connected to a ValveLink8.2 system, Automate Scientific, Berkeley, CA) and perfused at a rate
of 2.0 ml/min at room temperature with frog Ringer's solution (115 mM NaCl, 2.5 mM KCl, 10
mM HEPES, 1.8 mM CaCl2, pH 7.3) containing 1 μM atropine sulfate (Sigma-Aldrich) to block
potential muscarinic responses. The two-microelectrode voltage-clamp technique was used to
measure current responses at a constant holding potential (-60 mV for α7 and -50 mV for α4β2
nAChRs). The voltage-measuring microelectrode (filled with 3M KCl solution) resistance was

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0.5-3.0 MΩ and the current-passing electrode (containing 250 mM CsCl and 100 µM EGTA)
resistance was 0.5-2.0 MΩ. Membrane currents were recorded with an AxoClamp-2 (Axon
Instruments, Union City, CA). Sampling rates were between 5 and 10 Hz. Compounds were
transiently applied (2 sec for α7 and 5 sec for α4β2 receptors) to avoid a cumulative
desensitization. A rapid flow rate (2.0 ml/sec) permitted complete replacement of the fluid
bathing the oocyte at approximately 3X per second. Initially each oocyte received two control
applications of a near maximal stimulatory concentration of ACh (1 mM ACh for α7 and 100
µM ACh for α4β2 receptors) to obtain a consistent response. ACh control applications
alternated with test compound applications every 5 min. The peak current response for a given
compound concentration was then normalized with respect to the mean current response obtained
by averaging the responses for the ACh applications before and after that test concentration.
Clampfit 8.1 (Axon Instruments) was used for data acquisition and Prism 3.0 (GraphPad, San
Diego, CA, USA) for analysis. The concentration-response curve for each compound was
calculated by fitting the data using a modified Hill equation:
Response I = Imax (Agonist) / [(Agonist)n+(EC50)n]
where each Response current I is normalized with respect to the above-mentioned mean ACh
control response and Imax denotes the maximal response current for the agonist, again relative to
the ACh control response, and n is the Hill slope. It has been demonstrated that α7 nAChR total
current (net charge over time)-concentration response curves are shifted to lower concentrations
relative to transient peak response-concentration curves (Papke and Thinschmidt, 1998). Thus,
we measured net charge as well as peak current responses for each oocyte response. Total
current over a 20 s interval (including the 2 s drug application period) was measured and the net
charge response was obtained after subtraction of baseline current for the same 20 s period.

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Compound comparisons using the net charge method led to the same conclusions as for the peak
current method (Table 1). The number of oocytes (n) tested at each concentration was ≥4 and is
given in Figure 2. Efficacy (Imax) estimates by peak current and net charge methods and potency
(EC50) estimates are presented in Tables 1 and 3 as arithmetic means ± one sample standard
deviation. One way ANOVA of log10 transformed compound Imax and EC50 data was used to
compare all analogs with nicotine, with Dunnett’s multiple comparison post-test. Student’s two-
tailed t test was used to assess statistical significance between mean Imax and EC50 estimates for
the two enantiomers of a compound.
Radioligand binding experiments
Rat brain membrane radioligand binding experiments were carried out essentially as
previously described (Kem et al., 2004). Frozen adult male Sprague-Dawley rat brains (Pel-
Freeze Biologicals, Rogers, AZ), after thawing on ice and being sliced into smaller pieces, were
homogenized with a 30 ml Wheaton glass homogenizing tube and pestle in ice cold binding
saline (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 50 mM Tris-TrisHCl buffer, pH
7.4). After the homogenate was centrifuged at 14,600 x G for 10 min the resulting pellet was
resuspended in fresh binding saline, homogenized, centrifuged again and the supernatant
discarded. A protein assay (BCA, Pierce, Rockford, IL) was then performed to obtain the
protein concentration of the washed and pellet-resuspended membranes, which were stored at -
82C before use. Each tube in the binding experiment received 100 µg of rat brain membrane
protein.
Washed membranes from cultured TsA201 cells expressing human 42 nAChRs were
also utilized to assess the nAChR affinities of nicotine and most nicotine analogs. Although these

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cells are known to express both stoichiometries of this heteromeric receptor, nicotine binding
curves in our study were well fitted assuming a single population displaying the characteristics of
the highest affinity (4223) form (Kuryatov et al., 2005). Cells at 80-90% confluence were
collected with a disposable cell scraper after removing the culturing media from the flask (75
cm2) and adding 6-10 ml of ice-cold Tris binding saline. The dislodged cells were collected in
centrifuge tubes and spun down at 7,700 x G for 8 min. The loose pellet was collected and
homogenized as described above for rat brain membranes; after a protein assay the washed
membranes were stored at -85C before use. For TsA201 cell membranes, 50 g protein per
tube were used for radioligand binding experiments. SH-EP1 cells expressing human α7 nAChRs
were similarly cultured and their membranes (100 µg membrane protein per tube) were used in
125I--BTX displacement assays (Zhao et al., 2003).
Radioligands selective for each nAChR subtype were obtained from Perkin Elmer Life and
Analytical Sciences (Boston, MA). Using conditions reported to optimally measure α4β2 nAChR
affinity, 1.0 nM 3H-cytisine (34 Ci/mmol) displacement experiments were performed at 4oC as
previously reported by Pabreza et al. (1991). Measurements of α7 nAChR affinity were done by
displacement of 125I--BTX (136 Ci/mmol) binding; the final test 125I--BTX concentration was
0.32 nM. These experiments required incubation for three hours at 37C to assure equilibration.
Membranes were suspended in binding saline containing 2 mg/ml bovine serum albumin (Sigma-
Aldrich) to reduce non-specific binding. In each experiment, 48 disposable glass culture tubes
each (total volume 0.50 ml) containing equal concentrations of cell membranes and radioligand
but different concentrations of the compound of interest were always incubated together. For
each radioligand, nonspecific binding was measured in the presence of a final concentration of 1
mM nicotine hydrogen tartrate (Sigma-Aldrich). After incubation, radioligand bound to

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membranes in each tube was rapidly collected by vacuum filtration using a 48 position Brandel
cell harvester (Gaithersburg, MD) and Whatman GF/C glass fiber filters that were pre-soaked in
0.5% polyethylenimine (Sigma-Aldrich) for 45 minutes to reduce nonspecific binding. The
radiolabeled membranes were rapidly washed three times with 3 ml of ice-cold binding saline to
remove the unbound radioligand. Filters containing 3H-cytisine bound membranes were
collected in 20 ml scintillation tubes and suspended in 8 ml 30% Scintisafe (Fisher Scientific,
Atlanta, GA) scintillation fluid, then counted in a Beckman LS-6500 liquid scintillation counter
after standing overnight. Filters containing 125I--Btx bound membranes were placed in 4 ml
gamma vials and counted in a Beckman Instruments (Fullerton, CA) 5500B gamma counter.
Displacement assay binding data were analyzed using Prism software. The mean counts
per minute for each compound concentration were obtained from 4 replicates; the data was fitted
to a sigmoidal concentration response curve for one site binding, from which the Hill slope (n)
and IC50 were estimated as follows:
n
Y = Bottom + (Top-Bottom) / (1+10(X-Log IC50)
)
Top (of the curve) is the maximal specific binding plateau of radioligand, Bottom (of the curve)
is the minimum specific binding plateau observed at high concentrations of the displacing ligand
and X=Log (Compound), The Kd estimate for each radioligand and receptor, previously
calculated from saturation binding experiments carried out using the same incubation conditions,
was then used to calculate the equilibrium dissociation constant (Ki) value of the displacing
ligand from the Cheng-Prusoff equation: Ki = IC50/[1+(Radioligand)/Kd]. For [3H]-cytisine, the
binding Kd was 0.92 nM for rat brain membranes (RBM) and 0.48 nM for human α4β2 receptors
expressed in TsA201 cell membranes. For 125I--Btx, the Kd was 0.67 nM for RBM and 0.97

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nM for human α7 receptors expressed in SH-EP1 cell membranes. One way ANOVA of log10
transformed compound mean Kis obtained from a number of individual experiments was used to
compare analogs with nicotine, with Dunnett’s multiple comparison post-test. A Student’s two-
tailed t test was used to assess statistical significance between the mean Ki estimates for cis and
trans enantiomers of a compound.
Computer docking of nicotine analogs with nAChR model proteins
The protein crystal structures used for the in silico molecular docking were the Lymnaea
Acetylcholine Binding Protein (AChBP) occupied by nicotine (Celie et al., 2004; Brookhaven
Protein Data Bank accession code 1UW6) and a chimeric protein based on Aplysia californica
AChBP containing binding site mutations to better resemble α7 nAChR, which was occupied by
epibatidine. (Li et al., 2011, Brookhaven Protein Data Bank accession code 3SQ6). The
molecular docking of all nicotine analogs was preceded by a two step optimization process
aimed at obtaining consistent starting geometries. The random walk algorithm implemented in
HyperChem v8.0 (HyperCube Inc., Gainesville, FL) was used to sample the potential energy
surface to identify the lowest energy conformation of each molecule. One thousand
conformations per molecule were generated by varying randomly all dihedral angles and using
an acceptance energy cut-off of 6 kcal/mol above the best. All conformations with an energy
difference of less than 0.05 kcal/mol were considered duplicates and hence only one was
retained. Conformations with potentially “bad” (distance between any two atoms of less than
0.5Å) van der Waals contacts were discarded. The lowest energy conformation was further
subjected to a QM optimization in GAMESS (Schmidt et al., 1993) using an STO-3G basis set to
represent the wavefunction in the Hartree-Fock approximation. Molecular surface calculations

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for each nicotine analog were made using MSROLL (Dock 6.5 Manual, UCSF, San Francisco,
CA). Each molecular surface was used as input for the sphere generating program SPHGEN
(Dock 6.5, UCSF). A cluster of 77 spheres that overlapped with the ligand (a nicotine or
epibatidine molecule in the appropriate crystal structure) was manually selected to be the point of
interest in the subsequent docking calculations. SHOWBOX was used to construct a 3-
dimensional rectangle 6 Å in any direction from the sphere cluster. CHIMERA (Pettersen et al.,
2004) was used to prepare each protein structure and convert the file type to the necessary Mol2
format. The box file was used as input for the GRID program which calculated the necessary
information concerning the steric and electrostatic environment within the area of the box in the
1UW6 and 3SQ6 protein structures.
The nicotine analogs used as inputs for docking were energy minimized. DOCK6.5 (Allen
et al., 2015) was then used to measure the predicted binding energies of the compounds within
the binding pocket designated by the spheres. With ligand flexibility allowed, each compound
was docked in a minimum of 20,000 orientations. Separate computations were done for docking
to each of the following structures for the 1UW6 Lymnaea AChBP: (1) the water-free site, (2)
the site containing persistent water LWA (Amiri et al., 2007) bound to the side chain oxygen on
the complimentary surface, (3) the site containing LWB binding to Tyr 192 (Amiri et al., 2007)
and finally, and (4) the site containing both of these waters. The fit of the lowest energy docked
structure of each docked nicotine compound relative to the 1UW6 nicotine-AChBP structure was
estimated by calculating the root mean square deviation for the 11 ring atoms of nicotine (See
Table S2). The Internet-available program Coote was used for these determinations. To allow
comparison of the compound free energies of binding (calculated from the observed binding
affinities of the compounds for the two human receptors) with the docking energies predicted for

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the two AChBPs, the predicted energies of the various analogs were first normalized with respect
to the predicted nicotine docking energy. Then the predicted docking energies of the compounds
were normalized with respect to the experimental free energy changes (calculated from their Kis)
associated with their binding to SH-EP1 cell expressed human α7 receptors (Table 2). The
experimental and predicted free energies (ΔGs) are presented in Table 5.
[Figure 2]
RESULTS
Concentration-response relationships for ACh, nicotine and the methylnicotines
We measured the functional properties of each methylnicotine, namely, the EC50 (inversely
related to potency) and Imax (maximal ACh-normalized current), using Xenopus oocytes
expressing human α7 or α4β2 nAChRs, to allow comparison of the compound with nicotine and
its enantiomer. The concentration-current response curves of these two nAChRs for ACh and
nicotine are shown in Figure S1. In addition, the same curve for nicotine, but without the
standard error bars, is included as a dashed line in each concentration-response curve in Figure 2
to allow visual comparison with the nicotine analog curve. Because the α7 receptor is much less
sensitive to ACh than the αβ42 receptor, the standard calibrating agonist was 1,000 µM ACh,
but for the α4β2 nAChR it was 100 µM ACh. The two calibrating ACh concentrations were
chosen to produce near-maximal responses that would not cause cumulative desensitization
during the experiment.
Our concentration-response data for α7 receptors (Fig. 2, Table 1) includes both the more
readily recorded peak current responses as well as currents integrated over the entire time of
agonist response (net charge method). Both types of data were obtained from the same response.

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When administered by standard methods of oocyte perfusion, including ours, the α7 peak current
response for an administered concentration of agonist does not reflect the maximum degree of
receptor activation possible with that agonist concentration, because receptor desensitization is
more rapid than the solution change: the peak current occurs before the desired concentration is
reached at the oocyte membrane (Papke and Thinschmidt, 1998). Nevertheless, the peak current
response should allow us to quantitatively compare the α7 response of a methylated nicotine
relative to that of its enantiomer or nicotine, since the diffusion limited rates of equilibration of
these compounds with the receptor should be nearly the same. In support of this assumption, net
charge responses obtained from the agonist pulses yielded the same conclusions as for the peak
current responses, except that the net charge potency (EC50) estimates were 2-3-fold smaller (See
PC/NC ratios in Table 1).
[Tables 1&2]
Another consideration in the interpretation of the Xenopus oocyte functional data obtained
with α4β2 nAChRs regards their subunit stoichiometry. Our functional measurements of the
methylnicotine effects were nearly complete when the co-expression of α4β2 nAChRs with
different stoichiometries and pharmacological properties was shown to be of general occurrence
(Moroni et al., 2006). The α43β22 low ACh-nicotine sensitivity receptor was reported to have a
nicotine EC50 that is ~50X higher than for the high sensitivity α42β43 receptor (Tavares et al.,
2012). In our Xenopus oocyte experiments we routinely injected equal amounts of the two
mRNAs. The nicotine concentration-response curve (Figure S1) that we obtained for α4β2
nAChRs was fitted well with Prism assuming a single population of α4β2 nAChRs. When the
α4β2 mRNA ratio was either 4:1 or 1:4, we still obtained EC50 values for nicotine that were
almost the same as when the mRNA ratio was 1:1 (Results not shown). The Imax value of nicotine

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obtained with the 1:1 mRNA injected oocytes was nearly identical with that of the 100 µM ACh
response, which was ~60% of the Imax for ACh. In published studies where α4 and β2 cDNAs
were injected into the nucleus, the Imax for the high affinity form was 30-60% of the maximal
ACh current response (Moroni et al., 2006). Thus, our Imax values for α4β2 receptors are
consistent with previously published values for Imax obtained with the high affinity α42β23
receptor. Our more recent experiments co-injecting an α4β2 concatamer mRNA with the β2
mRNA, which should yield only α42β23 -like functional receptors (Fig S2), indicate that the near
maximal concatameric responses for several key analogs were very similar to the maximum
responses in our routine Xenopus experiments (Fig. S3), further supporting our contention that
the latter primarily assessed the properties of the high affinity stoichiometry α42β23 subtype.
The radioligand binding data measuring ligand affinity for α4β2 AChRs are unlikely to be
affected by the presence of the low affinity α43β22 receptors, since we measured displacement at
such a low concentration (1 nM) of [3H]-cytisine that few of these receptors would be occupied
(Tavares et al., 2012; Moroni et al., 2006).
The variance estimates for the mean potencies (EC50) and binding constants (Kis) of the
compounds tended to increase as their mean values increased, so we used a log10 transformation
of the estimates to better approximate a normal distribution of the individual estimates for a
compound. This resulted in more similar variance estimates among the compounds and allowed
one way ANOVA to be used to analyze comparisons of all compounds with nicotine.
Functional and radioligand binding data for each methylnicotine will now be presented and
discussed in order of the five pyrrolidinium ring atoms 1’ to 5’ (Figure 1) numbered according to
the International Union for Pure and Applied Chemistry nomenclature. Data for receptor α7

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nAChRs will be presented initially since interactions of these compounds with this receptor
subtype have not been previously reported.
[Tables 3 and 4]
1’-Methylnicotine and 1’-Ethylnornicotine
Ample evidence exists that the monocationic form of nicotine and of most other ionizable
nAChR agonists is the form which binds to nAChRs with highest affinity (Barlow and Hamilton,
1962; Jeng and Cohen, 1980; Kem et al., 2004). Since the published pKa for ionization of the
1’N of nicotine (8.05, Fujita et al., 1971) is 0.65 units above physiological pH of 7.4 and the
methylnicotine analogs were found to have similar pKas (Table S1), our pharmacological data in
Tables 1-6 largely reflect the properties of the monocationic species and are uncorrected for the
small differences in ionization we observed for the 1’-ethyl-, 2’-methyl- and 5’-methylnicotine
analogs (See Table S1 for estimation of the % monocationic species for compounds expected to
show the largest differences in ionization).
We first examined the consequences of adding an additional methyl substituent to the 1’-N
of nicotine and found major differences between the two receptors. For α7 receptors the agonist
properties (Figure 2, Table 1) and binding affinities (Table 2) of the quaternary nitrogen analog,
1’-methylnicotinium iodide, were similar to those of nicotine, but they were diminished at α4β2
receptors (Figure 2, Tables 3 and 4). Thus, methyl quaternization of the pyrrolidinium nitrogen is
well tolerated by α7 but not α4β2 nAChRs.
Replacing the 1’N-methyl group of nicotine with a larger alkyl group, as in 1’ethyl-
nornicotine, also differentially affected agonistic properties at these two receptors. The ethyl
substituent was relatively well tolerated by the α7 receptor: only the Imax was statistically

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different from that of nicotine (Table 1). However, at α4β2 nAChRs 1’-ethyl-nornicotine
potency and efficacy was inferior to that of nicotine (Table 3). We also found that racemic
1’propyl-nornicotine displayed almost a thousand-fold decrease in potency relative to nicotine at
α4β2 receptors, in agreement with a previous study (Glassco et al., 1994) as well as a greatly
reduced potency at α7 receptors (Results not shown). Thus, nicotine interaction with α4β2
receptors is particularly sensitive to the presence of bulky quantitatively alkyl groups at the 1’N
position.
2’-Methylnicotines
Trans-2’-methylation produced unique changes relative to the other six carbon-methylated
nicotines, who displayed reduced agonist properties relative to nicotine. 2’methylnicotine
displayed agonist properties superior to nicotine at α7 receptors and was at least as active as
nicotine at α4β2 receptors. α7 nAChR potency and equilibrium binding affinity (Table 1) were
enhanced about 7fold for the human receptor in comparison with nicotine. At the α4β2 receptor,
2’-methylnicotine potency and Imax were not statistically different from nicotine (Table 3). The
small increase in binding affinity at the α4β2 receptor was only statistically significant for the
human receptor (Table 4). Our data for 2’-methylnicotine binding to rat brain α4β2 receptors
was consistent with the data of Wang et al. (1998) for racemic 2’-methylnicotine, taking into
consideration that our 2’-methyl(S)nicotine has a much higher (>40 fold) higher binding affinity
than 2’-methyl(R)nicotine (Kem, unpublished results).
3’-Methylnicotines
Cis-3’-methylnicotine displayed less potency (~3 fold at α7 and ~10 fold at α4β2
receptors) relative to nicotine (Figure 2, Tables 1 and 3). There was also a statistically significant

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(~2 fold) decrease in the α4β2 receptor Imax. Cis-3’-methylnicotine displayed less affinity for α7
(approximately 4 fold for rat and 8 fold for human) and α4β2 (approximately 200 fold less for rat
and 400-fold less for human) receptors relative to nicotine (Tables 2 and 4). While the affinity of
trans-3’methylnicotine for the α7 receptor was similar to that of nicotine, its affinity for α4β2
receptors was ≥30 fold less than for nicotine.
4’-Methylnicotines
At both receptors (especially the α7) the efficacies of both 4’-methylnicotines were greatly
reduced relative to nicotine (Figure 2, Tables 1 and 3). Whereas the potencies of the two
enantiomers were inferior to that of nicotine at α7 receptors, on the human α4β2 receptor the
potencies were not significantly different from that of nicotine. Relative binding affinities of the
two enantiomers at the two receptors differed: at both α7 receptors the cis enantiomer displayed
the highest affinity, but at rat and human α4β2 receptors the trans-enantiomer displayed the
highest affinity.
5’-Methylnicotines
Analysis of the pharmacological properties of cis-5’-methylnicotine revealed its limited
ability to interact with α4β2 receptors. Agonist potency was reduced >700 fold and Imax >100
fold (Table 3). The binding Ki’s for rat and human α4β2 receptors also were increased >2,000
fold and >700 fold, respectively (Table 4). Previous radioligand binding studies also indicated
that methylation at this position had detrimental effects on nicotine binding affinity for rat brain
high affinity (α4β2) receptors and that the binding Ki of trans-5’-methyl-nicotine was less
affected than that of the cis-form (Lin et al., 1994; Wang et al., 1998).

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While cis-5’-methylnicotine also displayed the greatest reduction in interaction with α7
receptors, trans-5’-methylnicotine was a relatively potent agonist, potency decreasing only 2-3
fold and Imax decreasing ~1.5 fold relative to nicotine (Table 1). Binding affinities for the human
and rat α7 nAChRs were only decreased ~3 fold relative to nicotine (Table 2).
[Figure 3]
Relationships between receptor affinity and receptor potency
Compound EC50 = Kd / (E+1), where Kd is the equilibrium dissociation constant for initial
binding to the inactive state and E is the equilibrium constant associated with receptor activation
(Auerbach et al., 2016). If Ki estimates are related to the Kd, then our EC50 estimates might show
some relationship to the Kis, at least for compounds displaying low E values. We plotted Ki
versus EC50 estimates for each alkylnicotine to determine whether they might be related (Figures
3A and C). We used EC50 estimates obtained with the net charge method of analysis for EC50
and Imax characterization of the functional properties of the human α7 receptor in this Figure.
Both the α7 and α4β2 plots showed a relatively strong correlation between these two variables
(Part A, α7 correlation coefficient r2 = 0.90; Part C, α4β2 r2 = 0.61). Since the α7 net charge
determined EC50s (Table 1) were ~3 fold less than the peak current EC50 estimates, using our
standard 2 sec agonist exposure (and ~10 fold less for a 20 second exposure, Kem, unpublished
results), the actual α7 receptor EC50 for each compound may be as much as 10 fold less than the
peak current EC50 reported in Table 1 and used in Part A of this Figure.
It was recently reported that there is a linear relationship between Log Imax and Log EC50
for agonists at the mouse neuromuscular nAChR--that high efficacy is directly correlated with
high potency (Auerbach, 2016). In spite of the differences in how efficacy was measured (single

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channel recording in the Auerbach et al. study and oocyte net charge response here, a similar
correlation was observed with our α7 data (Figure 3B, r2=0.67). In Figure 3B the Imax estimates
for the most efficaceous compounds (2’MeNic, 1’MeNic and Nic) are very similar, though their
EC50s differ considerably, which would not be predicted from the Auerbach equation; it seems
likely that desensitization may limit the Imaxs measured for these highly potent agonists under our
experimental conditions. In contrast to the α7 plot in 3B, the α4β2 plot showed much more
scatter when plotted in an identical fashion (Figure 3D, r2=0.24).
Modeling methylnicotine interaction with the molluscan ACh binding proteins
Since high resolution crystal structures of the two nAChRs are still unavailable, we
attempted to predict the binding poses and relative docking free energies of the various
methylnicotines using the Brookhaven Protein Data Bank Lymnaea AChBP-nicotine 1UW6
structure (Celie et al., 2004) and the mutated Aplysia californica AChBP-epibatidine 3SQ6
structure where the binding site was mutated to be similar to the α7 site (Li et al., 2011). In the
1UW6 structure the cis- side of the nicotine pyrrolidinium ring comes in closest proximity with
the AChBP Trp143 indole side chain and the 1’N proton is also very near (2.6 Å) the Trp143
peptide carbonyl group, and is assumed to form an H-bond with it (Celie et al., 2004). In
contrast, the 2’-carbon of nicotine is not in close contact with any non-water atoms in the
AChBP binding site. Actually, the 2’-methyl is oriented towards the disulfide bond between the
vicinyl half-cystines in the C-loop. The structures of the various methylnicotines (as
monocations) were energy-minimized and docked to both AChBPs after removing the
crystallized ligands from their respective ACh binding sites. The relative energies for docking
the various methyl-nicotines (Table 5) to both AChBP structures predicted many of the
differences in binding affinity that were observed, particularly when two of the most persistently

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bound water molecules in the Lymnaea AChBP-nicotine complex (Amiri et al., 2007) were
retained within the binding site during docking (See Table S2 for fit estimates). For the 3’- and
5’-methylnicotines the docking score of the cis-methyl enantiomer predicted a less favorable free
energy of binding relative to the trans enantiomer, and cis-5’-methylnicotine was predicted to
have the least favorable free energy of binding of all the methyl analogs to both AChBPs (Table
5). The nicotine compound predictions for the Aplysia AChBP-α7 chimera binding site,
containing no water molecules, were similar to those obtained for the Lymnaea AChBP.
Comparison of free energy changes associated with compound binding to human nAChRs
with predicted free energy changes for binding to AChBPs
The ΔGs (Table 5) of the various analogs relative to the ΔG for nicotine binding at the
same receptor site, expressed within the parentheses as a percent change, are most salient. The
relative ΔG changes associated with methylation were much greater for α4β2 receptors than for
α7 receptors. The major exception was 2’-methylnicotine, which actually showed a greater free
energy decrease (lower Ki) relative to nicotine at the α7 receptor. This was only predicted for the
Aplysia AChBP-α7 chimera, whose orthosteric binding site most resembles the α7 binding site.
Another important observation was that the free energy of binding difference (-1.9 Kcal/mol) for
nicotine, between the human α4β2 receptor (ΔG = -10.8 Kcal/mol) and the α7 receptor (ΔG = -
8.9 Kcal/mol), almost disappeared in free energy comparisons for most other analogs other than
the 1’N-methynicotinium and 1’N-ethylnornicotine. This important observation will be discussed
later.
[Table 5]

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Influence of methyl substituent configuration on receptor interaction in other methylated
nicotine analogs
Abreo et al. (1996) synthesized and tested the nicotine analog A84543 in which an
oxymethylene group forms a bridge between the 3-pyridyl ring and the 2’(S) position of the
pyrrolidinium ring so that the pyridyl ring retains the same chirality as in nicotine. This
compound displayed very high affinity and potency at α4β2 receptors, exceeding that of nicotine,
indicating that this receptor not only tolerates a greater distance between the two nicotine ring N
atoms but binds this compound more readily than nicotine. To determine whether A84543 would
be similarly affected by 5’-methylation, we synthesized (See Supplement) and tested the two 5’-
methylA84543 diastereomers. The rat brain α4β2 receptor affinity (Table 6) of trans-5’-
methylA84543 was 7-fold higher than the cis-5’methyl-A84543. The rat brain high affinity
receptor Ki for A84543 has been reported to be 0.15±0.01 (Abreo et al., 1996) and 3.44±0.40 nM
(Ogunjirin et al., 2015) and the α7 receptor Ki was reported to be 340 ±50 nM (Ogunjirin et al.,
2015). While the difference we observed between the two methylated A84543 enantiomers was
approximately 10X less than it was for the 5’methyl-nicotines, it is clear that the preferential
binding of the trans-5’-methylnicotine also applies to trans-5’methyl A85443. Like A84543,
both methyl enantiomers interacted with α7 receptors with much lower affinity relative to α4β2
receptors, with the cis- enantiomer showing the least affinity and potency (Table 6). The
efficacies of trans-5’methyl-A84543 (Figure 4) at human α7 and α4β2 receptors were similar to
those of trans-5’methylnicotine (Tables 1 and 3).
Since we had prepared racemic 3’-and 5’methylnicotines and then separated the
diastereomeric (R)- and (S)-methylnicotines, this afforded the opportunity of determining
whether the relative or absolute configurations of these two methyl substituents are critical for

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optimal interaction with the receptors. Our binding data shows that at the 3’-position the same
relative configuration (trans) allows the highest affinity, irrespective of the pyridyl ring
substituent configuration. However, it is the absolute configuration of the 5’-methyl substituent
that determines relative activity, since the substituent in cis-5’methyl-(R)nicotine has the same
absolute configuration as the methyl in tran-5’methyl-(S)nicotine.
[Figure 4, Table 6]
DISCUSSION
Dominant role of the cationic N-alkyl moiety
Ionization of the most basic N atom is essential for efficient binding of nicotine and most
nicotinoids to various AChRs and AChBPs. The pyrrolidinyl N in nicotine has been reported to
possess a pKa of 8.05 and a pyridyl ring N pKa of 3.85 (Fujita et al., 1971). Thus, at
physiological pH there will be a mixture of the monocationic and unionized pyrrolidinyl forms
and their relative solution concentrations will depend upon the pH. Under the ionic strength
conditions of our radioligand binding measurements, the nicotine pKa1 was 8.00 and its
ionization was 80% at pH 7.4 (Table S1). The pKa1 of the pyrrolidinium N was enhanced the
most (to 8.44) by replacing the 1’N-methyl with an ethyl group. Methylating the 2’ or
5’positions produced very small increases in ionization at pH 7.4. Methylation at the 3’ or 4’
position would not be predicted to have a significant inductive effect on the pKa1.
Adding an additional methyl to the 1’N of nicotine greatly diminished interaction with the
α4β2 receptor without much effect on interaction with the α7 receptor. Substituting the larger
ethyl moiety for the 1’N-methyl group, as in 1’N-ethylnornicotine, was deleterious for
interaction with both receptors, especially the 42 receptor. Formation of the 1’N-Trp B

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carbonyl hydrogen bond seems to be important for nicotine binding to α4β2 receptors (Xiu et al.,
2009; Puskar et al., 2011). The high affinity of nicotine for the α4β2 receptor may at least
partially be due to this hydrogen bond, which seems less important for its interaction with the α7
nAChR subtype and may not be essential (Puskar et al., 2011; van Arnam et al., 2013). The
lesser effects on α7 interaction of these two 1’-N modifications suggests that a small increase in
bulk of the pyrrolidinium group does not adversely affect cation-π binding. The highly
detrimental influence of increasing the bulk of substituents at the 1’N for α4β2 receptor
interaction may be due to these bulky substituents interfering with formation of this hydrogen
bond.
2’Methylation uniquely increases interaction with the α7 receptor without adversely
affecting interaction with the α4β2 nAChR
Unexpectedly, methylation at the 2’-carbon, which also connects the two rings,
significantly enhances binding and agonist potency at the α7 nAChR without significantly
affecting interaction with the α4β2 nAChR. The 2’methyl may form a hydrophobic interaction
with some group and that may favor formation of an H bond between the 1’NH+ and the
receptor, which otherwise seems to be absent. If binding site waters are present in the vicinity of
the 1’NH of nicotine, they might be displaced by a methyl substituent like the 2’methyl (Barratt
et al., 2006; Leung et al., 2012). Water molecules are present in the agonist-bound AChBP as
well as in the agonist-free state of the AChBP binding site (Celie et al., 2004; Amiri et al., 2007)
and it has been found (Forli and Olson, 2011; this study) that better docking predictions can be
obtained when persistent water molecules observed in the nicotine-AChBP crystal binding site
are included in the computer docking. The actual basis for the potentiating effect of the 2’methyl
substituent can only be determined by additional experimental and computational studies.

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3’Cis-methylation reduces agonist activity more than 3’trans-methylation
Since the pyrrolidinium 3’carbon is next to the inter-ring bonding 2’carbon, it was
predicted that its methylation would alter the energy profile for inter-ring torsion and might even
prevent the compound from attaining an optimal conformation for agonistic binding and to the
receptor. While both 3’-methylnicotines were less active than nicotine, we found that that trans-
3’-methylnicotine was significantly more active than cis-3’-methylnicotine at both receptors.
The thermodynamically preferred inter-ring angles for cis-3’- and trans-3’-methylnicotine and
for nicotine are predicted to be very similar, but the energy barrier for rotation around the C2’-
C3 bond is much higher (60 Kcal/mol) for cis-3’-methylnicotine compared with 14 Kcal/mol for
trans-3’-methylnicotine (Seeman, 1984). One possibility is that the two rings must be able to
move with respect to each other in the binding or receptor activation processes, and the presence
of the 3’methyl substituent to varying degrees inhibits these movements. The data in Table 6,
indicating that the relative configuration of the 3’-methyl group is more important for activity
and binding than its absolute configuration, is consistent with this interpretation.
5’Cis-methylation causes a drastic loss of activity at both nAChRs
The 5’ position of the pyrrolidinium ring is adjacent to the 1’N hydrogen, which H-bonds
to the peptide carbonyl of Trp 143 at the Lymnaea AChBP and by inference at Trp B of the α4β2
nAChR (Xiu et al., 2009; Blum et al., 2010). The H-bond does not seem to be present or very
strong at α7 receptors, at least for ACh, epibatidine and varenicline (van Arnum et al., 2013).
Since the 5’cis-hydrogen of nicotine abuts the Trp 143 side chain in AChBP, we suggest that cis-
methylation at this site significantly lowers the free energy change associated with nicotine’s H-
bonding to the equivalent Trp B (residue 149) in the α4β2 receptor. Theoretical calculations of
the positive charge density of the various carbon atoms in the pyrrolidinium ring of nicotine

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indicate that the positive charge is less centered on the pyrrolidinyl N than on the three C atoms
(1’, 2’ and 5’) attached to the nicotine 1’N (Elmore and Dougherty, 2002). Regardless of
mechanism, the significant dimunition in 5’cis-methyl nicotine binding to both receptors
indicates that the 5’- carbon occupies a very critical position that can exert much control of
nicotine’s interaction with these receptors. The superior activity of 5’trans-methylA84543
relative to 5’cis-methylA84543 (Table 6 and Figure 4) is consistent with the notion that the
binding orientation this compound, though it contains an oxymethylene bridge between the
pyridyl and pyrrolidinyl rings, is very similar to that of nicotine. Our data regarding the relative
affinities of the 5’methyl(R)nicotine enantiomers indicates that it is the absolute configuration of
the 5’methyl group that determines the relative affinities of these enantiomers for both receptors.
The effects of 5’methylation were greatest on nicotine’s interaction with the α4β2 nAChR
receptor. Based on the earlier radioligand binding studies with some of these nicotine analogs on
α4β2 receptors, we anticipated that all methylations would diminish nicotine’s agonistic
properties. Our radioligand binding data demonstrated a preferential reduction in binding of the
3’cis- and 5’cis-methylated nicotines on both receptors, as well as for cis-4’-methylnicotine
binding to the rat α4β2 (but not the α7 receptor). While the molecular basis for this behavior is
not yet known, it suggests that interaction of the cis side of the pyrrolidinium ring with a part of
the receptor is of paramount importance. It seems likely, based on the close proximity of the cis-
side of the pyrrolidinium ring to Trp 143 in AChBPs and Trp B in the α4β2 x-ray structure
(Perez et al., 2016), that cis methylation generally interfers with nicotine’s interaction with this
component of the aromatic “box”. The lesser influence of cis-methylation on interaction with the
α7 receptor could be due to the lesser importance of 1’NH+ hydrogen-bonding to Trp B and/or